PROJECT SUMMARY/ABSTRACT In eukaryotes, genomic DNA is condensed into chromatin by associating with histone proteins, and the folding of chromatin organizes the genome within the cell nucleus and plays an essential role in regulating gene transcription. Chromatin dysfunction and the resulting dysregulation of gene expression is a well-recognized contributor to many human diseases, including cancers and neurological disorders. Despite decades of research and its crucial role in the expression of genes, the native chromatin structure of coding and regulatory regions of the genome remains poorly resolved. While conventional structural biology methods have yielded many high- resolution structures of chromatin components, a general limitation of these studies is that the conditions used to prepare samples in vitro do not faithfully recapitulate the native chromatin environment in vivo, due to complex and variable composition of native chromatin. On the other hand, methods that have been geared towards probing the native chromatin landscape in situ are well-suited to provide information of a broad scale (e.g. genome wide) but are limited in resolution or dimensionality. I aim to bridge the gap between these traditional approaches by analyzing the composition and structure of natively assembled, yet purified, chromatin of specific yeast gene loci. The initial phase of this work will be aimed at optimizing the preparation of isolated chromosomal fragments in order to maximize preservation of their native composition and structure, using a combination of electron microscopy (EM) and mass spectrometry proteomics as quality control assays. Quantitative mass spectrometry of isotope-labeled samples will also be used to perform ?chromatin proteomics? by analyzing changes in chromatin composition between the induced and repressed forms of the minichromosome genes. Following optimization of purification conditions, negative stain and rotary shadowing EM will be used to generate high-resolution two-dimensional maps of nucleosome positioning across populations of a given purified yeast gene in its active or repressed state. Finally, utilizing state-of-the-art instrumentation available at Johns Hopkins University, modern cryo-EM and cryo-electron tomography techniques will be used to analyze the three- dimensional chromatin structure of the purified gene, including an assessment of the variability in chromatin structure amongst populations of repressed or induced genes. Ultimately, this approach should provide an overview of the three-dimensional ?structure? of a gene in both repressed and activated states, as well as ?zoomed-in? views of specific gene fragments, showing for instance the interplay between histones and non- histone proteins at the promoter region of genes. The results of this study will significantly advance our understanding of the mechanisms behind transcriptional regulation via chromatin, and could provide insights into mechanisms of transcriptional dysregulation that cause disease.